Deuterated CFTR potentiators

ABSTRACT

This invention relates to compounds of Formula I: 
                         
and pharmaceutically acceptable salts thereof. This invention also provides compositions comprising a compound of this invention and the use of such compositions in methods of treating diseases and conditions that are beneficially treated by administering a CFTR potentiator.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/358,407, filed Nov. 22, 2016; which is a continuation of U.S.application Ser. No. 14/921,158, filed Oct. 23, 2015; which is acontinuation of U.S. application Ser. No. 14/490,787, filed Sep. 19,2014; which is a continuation of U.S. application Ser. No. 14/082,043,filed Nov. 18, 2013; which claims the benefit of U.S. ProvisionalApplication No. 61/860,602, filed Jul. 31, 2013; U.S. ProvisionalApplication No. 61/780,681, filed Mar. 13, 2013; and. U.S. ProvisionalApplication No. 61/727,941, filed Nov. 19, 2012. U.S. application Ser.No. 14/082,043 is also a continuation-in-part of PCT Application No.PCT/US2012/038297, filed May 17, 2012, which claims the benefit of U.S.Provisional Application No. 61/487,497, filed May 18, 2011. The contentsof each of these applications are incorporated herein by reference intheir entirety.

BACKGROUND OF THE INVENTION

Many current medicines suffer from poor absorption, distribution,metabolism and/or excretion (ADME) properties that prevent their wideruse or limit their use in certain indications. Poor ADME properties arealso a major reason for the failure of drug candidates in clinicaltrials. While formulation technologies and prodrug strategies can beemployed in some cases to improve certain ADME properties, theseapproaches often fail to address the underlying ADME problems that existfor many drugs and drug candidates. One such problem is rapid metabolismthat causes a number of drugs, which otherwise would be highly effectivein treating a disease, to be cleared too rapidly from the body. Apossible solution to rapid drug clearance is frequent or high dosing toattain a sufficiently high plasma level of drug. This, however,introduces a number of potential treatment problems such as poor patientcompliance with the dosing regimen, side effects that become more acutewith higher doses, and increased cost of treatment. A rapidlymetabolized drug may also expose patients to undesirable toxic orreactive metabolites.

Another ADME limitation that affects many medicines is the formation oftoxic or biologically reactive metabolites. As a result, some patientsreceiving the drug may experience toxicities, or the safe dosing of suchdrugs may be limited such that patients receive a suboptimal amount ofthe active agent. In certain cases, modifying dosing intervals orformulation approaches can help to reduce clinical adverse effects, butoften the formation of such undesirable metabolites is intrinsic to themetabolism of the compound.

In some select cases, a metabolic inhibitor will be co-administered witha drug that is cleared too rapidly. Such is the case with the proteaseinhibitor class of drugs that are used to treat HIV infection. The FDArecommends that these drugs be co-dosed with ritonavir, an inhibitor ofcytochrome P450 enzyme 3A4 (CYP3A4), the enzyme typically responsiblefor their metabolism (see Kempf, D. J. et al., Antimicrobial agents andchemotherapy, 1997, 41(3): 654-60). Ritonavir, however, causes adverseeffects and adds to the pill burden for HIV patients who must alreadytake a combination of different drugs. Similarly, the CYP2D6 inhibitorquinidine has been added to dextromethorphan for the purpose of reducingrapid CYP2D6 metabolism of dextromethorphan in a treatment ofpseudobulbar affect. Quinidine, however, has unwanted side effects thatgreatly limit its use in potential combination therapy (see Wang, L etal., Clinical Pharmacology and Therapeutics, 1994, 56(6 Pt 1): 659-67;and FDA label for quinidine at www.accessdata.fda.gov).

In general, combining drugs with cytochrome P450 inhibitors is not asatisfactory strategy for decreasing drug clearance. The inhibition of aCYP enzyme's activity can affect the metabolism and clearance of otherdrugs metabolized by that same enzyme. CYP inhibition can cause otherdrugs to accumulate in the body to toxic levels.

A potentially attractive strategy for improving a drug's metabolicproperties is deuterium modification. In this approach, one attempts toslow the CYP-mediated metabolism of a drug or to reduce the formation ofundesirable metabolites by replacing one or more hydrogen atoms withdeuterium atoms. Deuterium is a safe, stable, non-radioactive isotope ofhydrogen. Compared to hydrogen, deuterium forms stronger bonds withcarbon. In select cases, the increased bond strength imparted bydeuterium can positively impact the ADME properties of a drug, creatingthe potential for improved drug efficacy, safety, and/or tolerability.At the same time, because the size and shape of deuterium areessentially identical to those of hydrogen, replacement of hydrogen bydeuterium would not be expected to affect the biochemical potency andselectivity of the drug as compared to the original chemical entity thatcontains only hydrogen.

Over the past 35 years, the effects of deuterium substitution on therate of metabolism have been reported for a very small percentage ofapproved drugs (see, e.g., Blake, M I et al, J Pharm Sci, 1975,64:367-91; Foster, A B, Adv Drug Res, 1985, 14:1-40 (“Foster”); Kushner,D J et al, Can J Physiol Pharmacol, 1999, 79-88; Fisher, M B et al, CurrOpin Drug Discov Devel, 2006, 9:101-09 (“Fisher”)). The results havebeen variable and unpredictable. For some compounds deuteration causeddecreased metabolic clearance in vivo. For others, there was no changein metabolism. Still others demonstrated increased metabolic clearance.The variability in deuterium effects has also led experts to question ordismiss deuterium modification as a viable drug design strategy forinhibiting adverse metabolism (see Foster at p. 35 and Fisher at p.101).

The effects of deuterium modification on a drug's metabolic propertiesare not predictable even when deuterium atoms are incorporated at knownsites of metabolism. Only by actually preparing and testing a deuterateddrug can one determine if and how the rate of metabolism will differfrom that of its non-deuterated counterpart. See, for example, Fukuto etal. (J. Med. Chem., 1991, 34, 2871-76). Many drugs have multiple siteswhere metabolism is possible. The site(s) where deuterium substitutionis required and the extent of deuteration necessary to see an effect onmetabolism, if any, will be different for each drug.

This invention relates to novel derivatives of ivacaftor, andpharmaceutically acceptable salts thereof. This invention also providescompositions comprising a compound of this invention and the use of suchcompositions in methods of treating diseases and conditions that arebeneficially treated by administering a CFTR (cystic fibrosistransmembrane conductance regulator) potentiator.

Ivacaftor, also known as VX-770 and by the chemical name,N-(2,4-di-tert-butyl-5-hydroxyphenyl)-4-oxo-1,4-dihydroquinoline-3-carboxamide,acts as a CFTR potentiator. Results from phase III trials of VX-770 inpatients with cystic fibrosis carrying at least one copy of theG551D-CFTR mutation demonstrated marked levels of improvement in lungfunction and other key indicators of the disease including sweatchloride levels, likelihood of pulmonary exacerbations and body weight.VX-770 is also currently in phase II clinical trials in combination withVX-809 (a CFTR corrector) for the oral treatment of cystic fibrosispatients who carry the more common ΔF508-CFTR mutation. VX-770 wasgranted fast track designation and orphan drug designation by the FDA in2006 and 2007, respectively.

Despite the beneficial activities of VX-770, there is a continuing needfor new compounds to treat the aforementioned diseases and conditions.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the percentage of compound remaining over time forCompound 110 of the invention and for ivacaftor in human cytochromeP450-specific SUPERSOMES™.

DEFINITIONS

The term “treat” means decrease, suppress, attenuate, diminish, arrest,or stabilize the development or progression of a disease (e.g., adisease or disorder delineated herein), lessen the severity of thedisease or improve the symptoms associated with the disease.

“Disease” means any condition or disorder that damages or interfereswith the normal function of a cell, tissue, or organ.

It will be recognized that some variation of natural isotopic abundanceoccurs in a synthesized compound depending upon the origin of chemicalmaterials used in the synthesis. Thus, a preparation of VX-770 willinherently contain small amounts of deuterated isotopologues. Theconcentration of naturally abundant stable hydrogen and carbon isotopes,notwithstanding this variation, is small and immaterial as compared tothe degree of stable isotopic substitution of compounds of thisinvention. See, for instance, Wada, E et al., Seikagaku, 1994, 66:15;Gannes, L Z et al., Comp Biochem Physiol Mol Integr Physiol, 1998,119:725.

In the compounds of this invention any atom not specifically designatedas a particular isotope is meant to represent any stable isotope of thatatom. Unless otherwise stated, when a position is designatedspecifically as “H” or “hydrogen”, the position is understood to havehydrogen at its natural abundance isotopic composition. Also unlessotherwise stated, when a position is designated specifically as “D” or“deuterium”, the position is understood to have deuterium at anabundance that is at least 3000 times greater than the natural abundanceof deuterium, which is 0.015% (i.e., at least 45% incorporation ofdeuterium).

The term “isotopic enrichment factor” as used herein means the ratiobetween the isotopic abundance and the natural abundance of a specifiedisotope.

In other embodiments, a compound of this invention has an isotopicenrichment factor for each designated deuterium atom of at least 3500(52.5% deuterium incorporation at each designated deuterium atom), atleast 4000 (60% deuterium incorporation), at least 4500 (67.5% deuteriumincorporation), at least 5000 (75% deuterium), at least 5500 (82.5%deuterium incorporation), at least 6000 (90% deuterium incorporation),at least 6333.3 (95% deuterium incorporation), at least 6466.7 (97%deuterium incorporation), at least 6600 (99% deuterium incorporation),or at least 6633.3 (99.5% deuterium incorporation).

The term “isotopologue” refers to a species in which the chemicalstructure differs from a specific compound of this invention only in theisotopic composition thereof.

The term “compound,” when referring to a compound of this invention,refers to a collection of molecules having an identical chemicalstructure, except that there may be isotopic variation among theconstituent atoms of the molecules. Thus, it will be clear to those ofskill in the art that a compound represented by a particular chemicalstructure containing indicated deuterium atoms, will also contain lesseramounts of isotopologues having hydrogen atoms at one or more of thedesignated deuterium positions in that structure. The relative amount ofsuch isotopologues in a compound of this invention will depend upon anumber of factors including the isotopic purity of deuterated reagentsused to make the compound and the efficiency of incorporation ofdeuterium in the various synthesis steps used to prepare the compound.However, as set forth above the relative amount of such isotopologues intoto will be less than 49.9% of the compound. In other embodiments, therelative amount of such isotopologues in toto will be less than 47.5%,less than 40%, less than 32.5%, less than 25%, less than 17.5%, lessthan 10%, less than 5%, less than 3%, less than 1%, or less than 0.5% ofthe compound.

The invention also provides salts of the compounds of the invention. Asalt of a compound of this invention is formed between an acid and abasic group of the compound, such as an amino functional group, or abase and an acidic group of the compound, such as a carboxyl functionalgroup. According to another embodiment, the compound is apharmaceutically acceptable acid addition salt.

The term “pharmaceutically acceptable,” as used herein, refers to acomponent that is, within the scope of sound medical judgment, suitablefor use in contact with the tissues of humans and other mammals withoutundue toxicity, irritation, allergic response and the like, and arecommensurate with a reasonable benefit/risk ratio. A “pharmaceuticallyacceptable salt” means any non-toxic salt that, upon administration to arecipient, is capable of providing, either directly or indirectly, acompound of this invention. A “pharmaceutically acceptable counterion”is an ionic portion of a salt that is not toxic when released from thesalt upon administration to a recipient.

Acids commonly employed to form pharmaceutically acceptable saltsinclude inorganic acids such as hydrogen bisulfide, hydrochloric acid,hydrobromic acid, hydroiodic acid, sulfuric acid and phosphoric acid, aswell as organic acids such as para-toluenesulfonic acid, salicylic acid,tartaric acid, bitartaric acid, ascorbic acid, maleic acid, besylicacid, fumaric acid, gluconic acid, glucuronic acid, formic acid,glutamic acid, methanesulfonic acid, ethanesulfonic acid,benzenesulfonic acid, lactic acid, oxalic acid, para-bromophenylsulfonicacid, carbonic acid, succinic acid, citric acid, benzoic acid and aceticacid, as well as related inorganic and organic acids. Suchpharmaceutically acceptable salts thus include sulfate, pyrosulfate,bisulfate, sulfite, bisulfite, phosphate, monohydrogenphosphate,dihydrogenphosphate, metaphosphate, pyrophosphate, chloride, bromide,iodide, acetate, propionate, decanoate, caprylate, acrylate, formate,isobutyrate, caprate, heptanoate, propiolate, oxalate, malonate,succinate, suberate, sebacate, fumarate, maleate, butyne-1,4-dioate,hexyne-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate,dinitrobenzoate, hydroxybenzoate, methoxybenzoate, phthalate,terephthalate, sulfonate, xylene sulfonate, phenylacetate,phenylpropionate, phenylbutyrate, citrate, lactate, β-hydroxybutyrate,glycolate, maleate, tartrate, methanesulfonate, propanesulfonate,naphthalene-1-sulfonate, naphthalene-2-sulfonate, mandelate and othersalts. In one embodiment, pharmaceutically acceptable acid additionsalts include those formed with mineral acids such as hydrochloric acidand hydrobromic acid, and especially those formed with organic acidssuch as maleic acid.

The term “stable compounds,” as used herein, refers to compounds whichpossess stability sufficient to allow for their manufacture and whichmaintain the integrity of the compound for a sufficient period of timeto be useful for the purposes detailed herein (e.g., formulation intotherapeutic products, intermediates for use in production of therapeuticcompounds, isolatable or storable intermediate compounds, treating adisease or condition responsive to therapeutic agents).

“D” and “d” both refer to deuterium. “Stereoisomer” refers to bothenantiomers and diastereomers. “Tert” and “t-” each refer to tertiary.“US” refers to the United States of America.

“Substituted with deuterium” refers to the replacement of one or morehydrogen atoms with a corresponding number of deuterium atoms.

Throughout this specification, a variable may be referred to generally(e.g., “each R”) or may be referred to specifically (e.g., R¹, R², R³,etc.). Unless otherwise indicated, when a variable is referred togenerally, it is meant to include all specific embodiments of thatparticular variable.

Therapeutic Compounds

The present invention provides a compound of Formula I:

or a pharmaceutically acceptable salt thereof, whereineach of X¹, X², X³, X⁴, X⁵, X⁶, and X⁷ is independently hydrogen ordeuterium;each of Y¹, Y², Y³, Y⁴, Y⁵, and Y⁶ is independently CH₃ or CD₃;provided that if each of Y, Y³, Y⁴, Y⁵, and Y⁶ is CH₃, then at least oneof X¹, X², X³, X⁴, X⁵, X⁶, and X⁷ is deuterium.

In one embodiment, X¹, X², X³, and X⁴ are the same. In one aspect ofthis embodiment, X⁶ and X⁷ are the same. In one aspect of thisembodiment, Y¹, Y², and Y³ are the same. In one aspect of thisembodiment, Y⁴, Y⁵, and Y⁶ are the same. In one example of this aspect,Y¹, Y², and Y³ are the same. In a more particular example, X⁶ and X⁷ arethe same.

In one embodiment, each of Y¹, Y², and Y³ is the same. In one aspect ofthis embodiment, each of Y⁴, Y⁵, and Y⁶ is the same. In one example ofthis aspect, X⁶ and X⁷ are the same.

In one embodiment, at least one of C(Y)(Y²)(Y³) and C(Y⁴)(Y⁵)(Y⁶) isC(CD₃)₃.

In one embodiment, Y¹, Y², and Y³ are CD₃. In one aspect of thisembodiment, Y⁴, Y⁵, and Y⁶ are CH₃. In another embodiment, Y⁴, Y⁵, andY⁶ are CD₃. In one aspect of this embodiment, Y¹, Y², and Y³ are CH₃. Inyet another embodiment, Y¹, Y², Y³, Y⁴, Y⁵, and Y⁶ are CD₃. In yetanother embodiment, Y¹, Y², Y³, Y⁴, Y⁵, and Y⁶ are CH₃. In one aspect ofany embodiment wherein Y¹, Y², and Y³ are CD₃, X⁶ is hydrogen. In oneexample of this aspect, X⁷ is hydrogen. In another example of thisaspect, X⁷ is deuterium. In one aspect of any embodiment wherein Y¹, Y²,and Y³ are CD₃, X⁶ is deuterium. In one example of this aspect, X⁷ ishydrogen. In another example of this aspect, X⁷ is deuterium. In oneaspect of the embodiment wherein Y¹, Y², and Y³ are CD₃ and X⁶ isdeuterium, the isotopic enrichment factor for X⁶ is at least 4000 (60%deuterium incorporation), such as at least 4500 (67.5% deuteriumincorporation), such as at least 5000 (75% deuterium), but not greaterthan 5500 (82.5% deuterium incorporation).

In one aspect of any embodiment wherein Y¹, Y², and Y³ are CH₃, Y⁴, Y⁵,and Y⁶ are CD₃ and X⁶ is hydrogen. In one example of this aspect, X⁷ ishydrogen. In another example of this aspect, X⁷ is deuterium. In oneaspect of any embodiment wherein Y¹, Y², and Y³ are CH₃, Y⁴, Y⁵, and Y⁶are CD₃ and X⁶ is deuterium. In one example of this aspect, X⁷ ishydrogen. In another example of this aspect, X⁷ is deuterium. In oneaspect of any embodiment wherein Y¹, Y², and Y³ are CD₃, Y⁴, Y⁵, and Y⁶are CD₃ and X⁶ is deuterium. In one example of this aspect, X⁷ ishydrogen. In another example of this aspect, X⁷ is deuterium. In oneaspect of the embodiment wherein Y¹, Y², and Y³ are CH₃, Y⁴, Y⁵, and Y⁶are CD₃, and X⁶ is deuterium, the isotopic enrichment factor for X⁶ isat least 4000 (60% deuterium incorporation), such as at least 4500(67.5% deuterium incorporation), such as at least 5000 (75% deuterium),but not greater than 5500 (82.5% deuterium incorporation).

In one aspect of the embodiment wherein Y¹, Y², Y³, Y⁴, Y⁵, and Y⁶ areCH₃, X⁶ is deuterium. In one aspect of the embodiment wherein Y¹, Y²,Y³, Y⁴, Y⁵, and Y⁶ are CH₃, and X⁶ is deuterium, the isotopic enrichmentfactor for X⁶ is at least 4000 (60% deuterium incorporation), such as atleast 4500 (67.5% deuterium incorporation), such as at least 5000 (75%deuterium), but not greater than 5500 (82.5% deuterium incorporation).

In one embodiment, each of Y⁴, Y⁵, and Y⁶ is the same. In one aspect ofthis embodiment, X⁶ and X⁷ are the same.

In one example of any of the foregoing embodiments, aspects or examples,X⁵ is hydrogen. In another example, X⁵ is deuterium.

In one embodiment, the compound of Formula I is any one of the compoundsof table 1,

TABLE 1 Cmpd # X¹ X² X³ X⁴ X⁵ X⁶ X⁷ Y¹ Y² Y³ Y⁴ Y⁵ Y⁶ 100 D D D D D D DCD₃ CD₃ CD₃ CD₃ CD₃ CD₃ 101 H H H H D H H CD₃ CD₃ CD₃ CD₃ CD₃ CD₃ 102 HH H H D H H CD₃ CD₃ CD₃ CH₃ CH₃ CH₃ 103 H H H H D H H CH₃ CH₃ CH₃ CD₃CD₃ CD₃ 104 H H H H D H H CH₃ CH₃ CH₃ CH₃ CH₃ CH₃ 105 H H H H H H H CD₃CD₃ CD₃ CD₃ CD₃ CD₃ 106 H H H H H H H CD₃ CD₃ CD₃ CH₃ CH₃ CH₃ 107 H H HH H H H CH₃ CH₃ CH₃ CD₃ CD₃ CD₃or a pharmaceutically acceptable salt thereof, wherein any atom notdesignated as deuterium is present at its natural isotopic abundance.

In one embodiment, the compound of Formula I is any one of the compoundsof table 2,

TABLE 2 Cmpd # X¹ X² X³ X⁴ X⁵ X⁶ X⁷ Y¹ Y² Y³ Y⁴ Y⁵ Y⁶ 110 H H H H H D DCD₃ CD₃ CD₃ CD₃ CD₃ CD₃ 111 H H H H D D D CD₃ CD₃ CD₃ CD₃ CD₃ CD₃ 112 HH H H D D D CD₃ CD₃ CD₃ CH₃ CH₃ CH₃ 113 H H H H D D D CH₃ CH₃ CH₃ CD₃CD₃ CD₃ 114 H H H H D D D CH₃ CH₃ CH₃ CH₃ CH₃ CH₃ 115 H H H H H D D CD₃CD₃ CD₃ CH₃ CH₃ CH₃ 116 H H H H H D D CH₃ CH₃ CH₃ CD₃ CD₃ CD₃ 117 D D DD D D D CD₃ CD₃ CD₃ CH₃ CH₃ CH₃ 118 D D D D D D D CH₃ CH₃ CH₃ CD₃ CD₃CD₃ 119 D D D D D H H CD₃ CD₃ CD₃ CH₃ CH₃ CH₃ 120 D D D D D H H CH₃ CH₃CH₃ CD₃ CD₃ CD₃ 121 H H H H H H D CD₃ CD₃ CD₃ CD₃ CD₃ CD₃ 122 H H H H HH D CD₃ CD₃ CD₃ CH₃ CH₃ CH₃ 123 H H H H H H D CH₃ CH₃ CH₃ CD₃ CD₃ CD₃124 H H H H H D H CD₃ CD₃ CD₃ CD₃ CD₃ CD₃ 125 H H H H H D H CD₃ CD₃ CD₃CH₃ CH₃ CH₃ 126 H H H H H D H CH₃ CH₃ CH₃ CD₃ CD₃ CD₃or a pharmaceutically acceptable salt thereof, wherein any atom notdesignated as deuterium is present at its natural isotopic abundance.

In one embodiment, the compound of Formula I is any one of the compoundsof table 3,

TABLE 3 Cmpd # X¹ X² X³ X⁴ X⁵ X⁶ X⁷ Y¹ Y² Y³ Y⁴ Y⁵ Y⁶ 301 D D D D D H HCD₃ CD₃ CD₃ CD₃ CD₃ CD₃ 302 D D D D D D D CH₃ CH₃ CH₃ CH₃ CH₃ CH₃ 303 DD D D D H H CH₃ CH₃ CH₃ CH₃ CH₃ CH₃ 304 D D D D H D D CD₃ CD₃ CD₃ CD₃CD₃ CD₃ 305 D D D D H H H CD₃ CD₃ CD₃ CD₃ CD₃ CD₃ 306 D D D D H D D CH₃CH₃ CH₃ CH₃ CH₃ CH₃ 307 D D D D H H H CH₃ CH₃ CH₃ CH₃ CH₃ CH₃ 308 H H HH H D D CH₃ CH₃ CH₃ CH₃ CH₃ CH₃or a pharmaceutically acceptable salt thereof, wherein any atom notdesignated as deuterium is present at its natural isotopic abundance.

In one embodiment, X⁷ is deuterium wherein the isotopic enrichmentfactor for X⁷ is between 66.7 (1% deuterium incorporation) and 1333.3(20% deuterium incorporation), such as between 333.3 (5% deuteriumincorporation) and 1000 (15% deuterium incorporation), such as between500 (7.5% deuterium incorporation) and 833.3 (12.5% deuteriumincorporation), such as 666.7 (10% deuterium incorporation) or as 733.3(11% deuterium incorporation). In one aspect of this embodiment, Y¹, Y²,and Y³ are each CH₃, Y⁴, Y⁵, and Y⁶ are each CD₃ and X⁶ is hydrogen. Inone aspect of this embodiment, Y¹, Y², and Y³ are each CH₃, Y⁴, Y⁵, andY⁶ are each CD₃ and X⁶ is deuterium. In one aspect of this embodiment,Y¹, Y², and Y³ are each CD₃, Y⁴, Y⁵, and Y⁶ are each CD₃ and X⁶ ishydrogen. In one aspect of this embodiment, Y¹, Y², and Y³ are each CD₃,Y⁴, Y⁵, and Y⁶ are each CD₃ and X⁶ is deuterium. In one aspect of thisembodiment, Y¹, Y², and Y³ are each CD₃, Y⁴, Y⁵, and Y⁶ are each CH₃ andX⁶ is hydrogen. In one aspect of this embodiment, Y¹, Y², and Y³ areeach CD₃, Y⁴, Y⁵, and Y⁶ are each CH₃ and X⁶ is deuterium.

In another set of embodiments, any atom not designated as deuterium inany of the embodiments, examples or aspects set forth above is presentat its natural isotopic abundance.

In another embodiment, the invention is directed to a compound selectedfrom the group consisting of:

or a salt thereof,wherein any atom not designated as deuterium is present at its naturalisotopic abundance.

In one aspect of compound 11c, 11c′, 12c, 13c, or 13c′, the percentageof isotopic enrichment at the CD ortho to the phenolic oxygen is about70%,

In one aspect of compound 11d, 12d, or 13d, the percentage of isotopicenrichment at the CD ortho to the OCOOCH₃ is about 70%,

In another embodiment, the invention is directed to a process comprisingone or more of the following:

-   -   i) treating 11a with a source of deuterium, such as DX, wherein        X is OH, OD or halo such as Cl, to form 11b;    -   ii) treating 11b with a source of —C(CD₃)₃, such as X—C(CD₃)₃,        wherein X is OH, OD or halo such as Cl, to form 11c;    -   iii) treating 11b with an acyloxcarbonylating agent, such as        X—C(O)OCH₃, wherein X is OH or halo such as Cl, to form 11d;    -   iv)        -   (I) treating 11d with a nitrating agent, such as HNO₃/H₂SO₄            to form a nitroaryl compound;        -   (II) treating the compound formed in (I) with a deprotecting            agent, such as sodium or potassium hydroxide in an alcohol            such as methanol to form a deprotected compound;        -   (III) treating the compound formed in (II) with a reducing            agent, such as ammonium formate and palladium over carbon to            form an aminoaryl compound;        -   (IV) treating the compound formed in (III) with a reducing            agent, with an acid such as HX, wherein X is halo such as            Cl, to form B″-1.

In another embodiment, the invention is directed to a process comprisingone or more of the following:

-   -   i. treating 12a with a source of —C(CD₃)₃, such as X—C(CD₃)₃,        wherein X is OH, OD or halo such as Cl, to form 12b;    -   ii. treating 12b with a source of —C(CH₃)₃, such as X—C(CH₃)₃,        wherein X is OH or halo such as Cl, to form 12c;    -   iii. treating 12c with an acyloxcarbonylating agent, such as        X—C(O)OCH₃, wherein X is OH or halo such as Cl, to form 12d;    -   iv.        -   I. treating 12d with a nitrating agent, such as HNO₃/H₂SO₄            to form a nitroaryl compound;        -   II. treating the compound formed in (I) with a deprotecting            agent, such as sodium or potassium hydroxide in an alcohol            such as methanol to form a deprotected compound;        -   III. treating the compound formed in (II) with a reducing            agent, such as ammonium formate and palladium over carbon to            form an aminoaryl compound;        -   IV. treating the compound formed in (III) with a reducing            agent, with an acid such as HX, wherein X is halo such as            Cl, to form B″-2.

In another embodiment, the invention is directed to a process comprisingone or more of the following:

-   -   i. treating 13a with a source of deuterium, such as DX, wherein        X is OH, OD or halo such as Cl, to form 13b;    -   ii. treating 13c with a source of —C(CD₃)₃, such as X—C(CD₃)₃,        wherein X is OH, OD or halo such as Cl, to form 13c;    -   iii. treating 13c with an acyloxcarbonylating agent, such as        X—C(O)OCH₃, wherein X is OH or halo such as Cl, to form 13d;    -   iv.        -   I. treating 13d with a nitrating agent, such as HNO₃/H₂SO₄            to form a nitroaryl compound;        -   II. treating the compound formed in (I) with a deprotecting            agent, such as sodium or potassium hydroxide in an alcohol            such as methanol to form a deprotected compound;        -   III. treating the compound formed in (II) with a reducing            agent, such as ammonium formate and palladium over carbon to            form an aminoaryl compound;        -   IV. treating the compound formed in (III) with a reducing            agent, with an acid such as HX, wherein X is halo such as            Cl, to form B″-3.

The synthesis of compounds of Formula I may be readily achieved bysynthetic chemists of ordinary skill by reference to the ExemplarySynthesis and Examples disclosed herein. Relevant procedures analogousto those of use for the preparation of compounds of Formula I andintermediates thereof are disclosed, for instance in WO 2007075946, WO2011019413, WO 2010019239, WO 2007134279, WO 2007079139 and WO2006002421, the teachings of which are incorporated herein by reference.

Such methods can be carried out utilizing corresponding deuterated andoptionally, other isotope-containing reagents and/or intermediates tosynthesize the compounds delineated herein, or invoking standardsynthetic protocols known in the art for introducing isotopic atoms to achemical structure.

Exemplary Synthesis

A convenient method for synthesizing compounds of Formula I is depictedin Scheme 1. Compounds of Formula I can be prepared wherein each D inthe CD3 group has an isotopic enrichment of at least 90% and preferablyat least 95%.

Compounds of Formula I may be prepared as shown in Scheme 1 via thecoupling of A and B employing HATU(N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)uroniumhexafluorophosphate) in the presence of DIEA(N,N′-diisopropylethylamine).

Deuterated intermediates of type A (scheme 1) may be prepared asoutlined in scheme 2, analogously to Singh, A.; Van Goor, F.; Worley, F.J. III; Knapp, T. Compounds Useful in CFTR Assays and Methods TherewithWO 2007075946 A1 Jul. 5, 2007, the entire teachings of which areincorporated herein by reference.

As shown in Scheme 2, heating a mixture of an aniline 1 in the presenceof a malonate derivative 2 affords the appropriately deuterated((phenylamino)methylene)malonate, subsequent exposure of which topolyphosphoric acid in the presence of POCl₃ followed by esterhydrolysis provides carboxylic acid A. In Scheme 2, X is hydrogen ordeuterium. In one embodiment, X, X¹, X², X³ and X⁴ are the same.

Exemplary compounds for use in Scheme 2 include the embodiment ofcompound (1) where X, X¹, X², X³ and X⁴ are each deuterium, commerciallyavailable from Aldrich; and the embodiment of compound (2) where X⁵ isdeuterium, prepared analogously to the procedure described in Scheme 2a(Parham, W. E.; Reed, L. J. Org. Syn., 1948, 28, 60, the teachings ofwhich are incorporated herein by reference) for the case where X⁵ ishydrogen by employing the embodiment of 3 where X⁵ is deuterium(available from CDN Isotopes). As shown in Scheme 2a, the appropriatelydeuterated (ethoxymethylene)malonate of type 2 may be prepared byreaction of diethylmalonate with the appropriately deuteratedtriethylorthoformate of type 3 in the presence of acetic anhydride andfacilitated by ZnCl₂.

Deuterated intermediates of type B (Scheme 1) may be prepared asoutlined in scheme 3, analogously to Singh, A. et al., supra.

As shown in Scheme 3, protection of di-tertbutylphenols of type 4 withmethyl chloroformate followed by exposure to nitric acid results in theformation of a nitro-methylcarbonate intermediate. Subsequent carbonatehydrolysis followed by palladium catalyzed reduction of the nitro groupultimately affords aminophenols of type B. In Scheme 3, X′ is hydrogenor deuterium. In one embodiment, X′, X⁶ and X⁷ are the same.

Compounds of formula B in Scheme 3 may be treated with either DCl or HClto obtain compounds of formula B′, or B″, respectively, with a highpercentage incorporation of deuterium or hydrogen, respectively, at theX⁶ position. This procedure is effective regardless of whether X⁶ in Bis hydrogen or deuterium. Thus, if X⁶ in B is hydrogen, or deuterium ata lower level of isotopic purity than desired, treatment with DClprovides B′; while if X⁶ is deuterium in B, treatment with HCl providesB″. Both treatments are shown in the two equations below:

The procedure may be used to exchange H for D, or D for H, and to enrichcompounds of formula B having lower levels of isotopic purity (0-85%) atX⁶. This procedure facilitates the preparation of compounds of formulaB′ or B″ containing >95% D or >95% H, respectively, at the positioncorresponding to X⁶. B′ or B″ may be then treated with A according toscheme 1 to afford a compound of Formula I wherein X⁶ is, respectively,deuterium or hydrogen.

Intermediates of the formula B″-1 may analogously be prepared inaccordance with the following scheme (where any atom not designated asdeuterium is present at its natural isotopic abundance):

Treatment of 11a with DCl in D₂O at 140° C. efficiently exchanges theortho hydrogens as well as the phenolic OH with deuterium providing 11b.Exposure of 11b to d10-t-butanol in the presence of D₂SO₄ selectivelyinstalls the d9-t-butyl moiety at the position ortho to the hydroxylgroup affording 11c. Reaction of 11c with methyl chloroformate provides11d which is then converted to B″-1 via a four step sequence involvingnitration, carbonate hydrolysis, nitro reduction and HCl mediated D to Hexchange.

Intermediates of the formula B″-2 may analogously be prepared inaccordance with the following scheme (where any atom not designated asdeuterium is present at its natural isotopic abundance):

Treatment of 12a with d9-t-butylchloride in the presence of ReBr(CO)₅selectively installs the d9-t-butyl moiety at the position para to thehydroxyl group affording 12b. Exposure of 12b to t-butanol in thepresence of H₂SO₄ selectively installs the t-butyl moiety at theposition ortho to the hydroxyl group affording 12c. Conversion of 12c toB″-2 follows the procedure described above for the conversion of 11c toB″-1.

Intermediates of the formula B″-3 may analogously be prepared inaccordance with the following scheme (where any atom not designated asdeuterium is present at its natural isotopic abundance):

Treatment of 13a with DCl in D₂O at 140° C. efficiently exchanges theortho and para hydrogens as well as the phenolic OH with deuteriumproviding 13b. Exposure of 13b to d10-t-butanol in the presence of D₂SO₄selectively installs the d9-t-butyl moieties at the 2 and 4 positionsaffording 13c. Conversion of 13c to B″-3 follows the procedure describedabove for the conversion of 11c to B″-1.

B″-1, B″-2 and B″-3 may be respectively converted to the correspondingcompounds of formula I by treatment with A analogously to the disclosureof Scheme 1.

Deuterated intermediates of type 4 (Scheme 3) may be prepared asoutlined in Schemes 4a-4d, analogously to Sun, Y.; Tang, N. Huaxue Shiji2004, 26, 266-268, the entire teachings of which are incorporated hereinby reference.

As shown in Schemes 4a-4d, ditertbutylphenols of type 4 may be preparedvia Friedel-Crafts alkylation of the appropriately deuterated phenol(phenol, 4-tert-butyl phenol or 2-tert butylphenol) with d9-tertbutylchloride. The embodiments of compound (4) that may be obtained asshown in scheme 4 are exemplary compounds for use in Scheme 3. In theembodiments 4a, 4b, 4c, and 4d in Scheme 4, any atom not designated asdeuterium is present at its natural isotopic abundance. In Scheme 4,both Compound 5 and compound 6 are commercially available (CDNIsotopes).

The specific approaches and compounds shown above are not intended to belimiting. The chemical structures in the schemes herein depict variablesthat are hereby defined commensurately with chemical group definitions(moieties, atoms, etc.) of the corresponding position in the compoundformulae herein, whether identified by the same variable name (i.e., R¹,R², R³, etc.) or not. The suitability of a chemical group in a compoundstructure for use in the synthesis of another compound is within theknowledge of one of ordinary skill in the art.

Additional methods of synthesizing compounds of Formula I and theirsynthetic precursors, including those within routes not explicitly shownin schemes herein, are within the means of chemists of ordinary skill inthe art. Synthetic chemistry transformations and protecting groupmethodologies (protection and deprotection) useful in synthesizing theapplicable compounds are known in the art and include, for example,those described in Larock R, Comprehensive Organic Transformations, VCHPublishers (1989); Greene, T W et al., Protective Groups in OrganicSynthesis, 3^(rd) Ed., John Wiley and Sons (1999); Fieser, L et al.,Fieser and Fieser's Reagents for Organic Synthesis, John Wiley and Sons(1994); and Paquette, L, ed., Encyclopedia of Reagents for OrganicSynthesis, John Wiley and Sons (1995) and subsequent editions thereof.

Combinations of substituents and variables envisioned by this inventionare only those that result in the formation of stable compounds.

Compositions

The invention also provides pharmaceutical compositions comprising aneffective amount of a compound of Formula I (e.g., including any of theformulae herein), or a pharmaceutically acceptable salt of saidcompound; and a pharmaceutically acceptable carrier. The carrier(s) are“acceptable” in the sense of being compatible with the other ingredientsof the formulation and, in the case of a pharmaceutically acceptablecarrier, not deleterious to the recipient thereof in an amount used inthe medicament.

Pharmaceutically acceptable carriers, adjuvants and vehicles that may beused in the pharmaceutical compositions of this invention include, butare not limited to, ion exchangers, alumina, aluminum stearate,lecithin, serum proteins, such as human serum albumin, buffer substancessuch as phosphates, glycine, sorbic acid, potassium sorbate, partialglyceride mixtures of saturated vegetable fatty acids, water, salts orelectrolytes, such as protamine sulfate, disodium hydrogen phosphate,potassium hydrogen phosphate, sodium chloride, zinc salts, colloidalsilica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-basedsubstances, polyethylene glycol, sodium carboxymethylcellulose,polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers,polyethylene glycol and wool fat.

If required, the solubility and bioavailability of the compounds of thepresent invention in pharmaceutical compositions may be enhanced bymethods well-known in the art. One method includes the use of lipidexcipients in the formulation. See “Oral Lipid-Based Formulations:Enhancing the Bioavailability of Poorly Water-Soluble Drugs (Drugs andthe Pharmaceutical Sciences),” David J. Hauss, ed. Informa Healthcare,2007; and “Role of Lipid Excipients in Modifying Oral and ParenteralDrug Delivery: Basic Principles and Biological Examples,” Kishor M.Wasan, ed. Wiley-Interscience, 2006.

Another known method of enhancing bioavailability is the use of anamorphous form of a compound of this invention optionally formulatedwith a poloxamer, such as LUTROL™ and PLURONIC™ (BASF Corporation), orblock copolymers of ethylene oxide and propylene oxide. See U.S. Pat.No. 7,014,866; and United States patent publications 20060094744 and20060079502.

The pharmaceutical compositions of the invention include those suitablefor oral, rectal, nasal, topical (including buccal and sublingual),vaginal or parenteral (including subcutaneous, intramuscular,intravenous and intradermal) administration. In certain embodiments, thecompound of the formulae herein is administered transdermally (e.g.,using a transdermal patch or iontophoretic techniques). Otherformulations may conveniently be presented in unit dosage form, e.g.,tablets, sustained release capsules, and in liposomes, and may beprepared by any methods well known in the art of pharmacy. See, forexample, Remington: The Science and Practice of Pharmacy, LippincottWilliams & Wilkins, Baltimore, Md. (20th ed. 2000).

Such preparative methods include the step of bringing into associationwith the molecule to be administered ingredients such as the carrierthat constitutes one or more accessory ingredients. In general, thecompositions are prepared by uniformly and intimately bringing intoassociation the active ingredients with liquid carriers, liposomes orfinely divided solid carriers, or both, and then, if necessary, shapingthe product.

In certain embodiments, the compound is administered orally.Compositions of the present invention suitable for oral administrationmay be presented as discrete units such as capsules, sachets, or tabletseach containing a predetermined amount of the active ingredient; apowder or granules; a solution or a suspension in an aqueous liquid or anon-aqueous liquid; an oil-in-water liquid emulsion; a water-in-oilliquid emulsion; packed in liposomes; or as a bolus, etc. Soft gelatincapsules can be useful for containing such suspensions, which maybeneficially increase the rate of compound absorption.

In the case of tablets for oral use, carriers that are commonly usedinclude lactose and corn starch. Lubricating agents, such as magnesiumstearate, are also typically added. For oral administration in a capsuleform, useful diluents include lactose and dried cornstarch. When aqueoussuspensions are administered orally, the active ingredient is combinedwith emulsifying and suspending agents. If desired, certain sweeteningand/or flavoring and/or coloring agents may be added.

Compositions suitable for oral administration include lozengescomprising the ingredients in a flavored basis, usually sucrose andacacia or tragacanth; and pastilles comprising the active ingredient inan inert basis such as gelatin and glycerin, or sucrose and acacia.

Compositions suitable for parenteral administration include aqueous andnon-aqueous sterile injection solutions which may contain anti-oxidants,buffers, bacteriostats and solutes which render the formulation isotonicwith the blood of the intended recipient; and aqueous and non-aqueoussterile suspensions which may include suspending agents and thickeningagents. The formulations may be presented in unit-dose or multi-dosecontainers, for example, sealed ampules and vials, and may be stored ina freeze dried (lyophilized) condition requiring only the addition ofthe sterile liquid carrier, for example water for injections,immediately prior to use. Extemporaneous injection solutions andsuspensions may be prepared from sterile powders, granules and tablets.

Such injection solutions may be in the form, for example, of a sterileinjectable aqueous or oleaginous suspension. This suspension may beformulated according to techniques known in the art using suitabledispersing or wetting agents (such as, for example, Tween 80) andsuspending agents. The sterile injectable preparation may also be asterile injectable solution or suspension in a non-toxicparenterally-acceptable diluent or solvent, for example, as a solutionin 1,3-butanediol. Among the acceptable vehicles and solvents that maybe employed are mannitol, water, Ringer's solution and isotonic sodiumchloride solution. In addition, sterile, fixed oils are conventionallyemployed as a solvent or suspending medium. For this purpose, any blandfixed oil may be employed including synthetic mono- or diglycerides.Fatty acids, such as oleic acid and its glyceride derivatives are usefulin the preparation of injectables, as are naturalpharmaceutically-acceptable oils, such as olive oil or castor oil,especially in their polyoxyethylated versions. These oil solutions orsuspensions may also contain a long-chain alcohol diluent or dispersant.

The pharmaceutical compositions of this invention may be administered inthe form of suppositories for rectal administration. These compositionscan be prepared by mixing a compound of this invention with a suitablenon-irritating excipient which is solid at room temperature but liquidat the rectal temperature and therefore will melt in the rectum torelease the active components. Such materials include, but are notlimited to, cocoa butter, beeswax and polyethylene glycols.

The pharmaceutical compositions of this invention may be administered bynasal aerosol or inhalation. Such compositions are prepared according totechniques well-known in the art of pharmaceutical formulation and maybe prepared as solutions in saline, employing benzyl alcohol or othersuitable preservatives, absorption promoters to enhance bioavailability,fluorocarbons, and/or other solubilizing or dispersing agents known inthe art. See, e.g.: Rabinowitz J D and Zaffaroni A C, U.S. Pat. No.6,803,031, assigned to Alexza Molecular Delivery Corporation.

Topical administration of the pharmaceutical compositions of thisinvention is especially useful when the desired treatment involves areasor organs readily accessible by topical application. For topicalapplication topically to the skin, the pharmaceutical composition shouldbe formulated with a suitable ointment containing the active componentssuspended or dissolved in a carrier. Carriers for topical administrationof the compounds of this invention include, but are not limited to,mineral oil, liquid petroleum, white petroleum, propylene glycol,polyoxyethylene polyoxypropylene compound, emulsifying wax, and water.Alternatively, the pharmaceutical composition can be formulated with asuitable lotion or cream containing the active compound suspended ordissolved in a carrier. Suitable carriers include, but are not limitedto, mineral oil, sorbitan monostearate, polysorbate 60, cetyl esterswax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol, and water. Thepharmaceutical compositions of this invention may also be topicallyapplied to the lower intestinal tract by rectal suppository formulationor in a suitable enema formulation. Topically-transdermal patches andiontophoretic administration are also included in this invention.

Application of the subject therapeutics may be local, so as to beadministered at the site of interest. Various techniques can be used forproviding the subject compositions at the site of interest, such asinjection, use of catheters, trocars, projectiles, pluronic gel, stents,sustained drug release polymers or other device which provides forinternal access.

In another embodiment, a composition of this invention further comprisesa second therapeutic agent. The second therapeutic agent may be selectedfrom any compound or therapeutic agent known to have or thatdemonstrates advantageous properties when administered with a compoundhaving the same mechanism of action as VX-770.

Preferably, the second therapeutic agent is an agent useful in thetreatment of a variety of conditions, including cystic fibrosis,Hereditary emphysema, Hereditary hemochromatosis,Coagulation-Fibrinolysis deficiencies, such as Protein C deficiency,Type 1 hereditary angioedema, Lipid processing deficiencies, such asFamilial hypercholesterolemia, Type 1 chylomicronemia,Abetalipoproteinemia, Lysosomal storage diseases, such as I-celldisease/Pseudo-Hurler, Mucopolysaccharidoses, Sandhof/Tay-Sachs,Crigler-Najjar type II, Polyendocrinopathy/Hyperinsulemia, Diabetesmellitus, Laron dwarfism, Myleoperoxidase deficiency, Primaryhypoparathyroidism, Melanoma, Glycanosis CDG type 1, Hereditaryemphysema, Congenital hyperthyroidism, Osteogenesis imperfecta,Hereditary hypofibrinogenemia, ACT deficiency, Diabetes insipidus (DI),Neurophyseal DI, Neprogenic DI, Charcot-Marie Tooth syndrome,Perlizaeus-Merzbacher disease, neurodegenerative diseases such asAlzheimer's disease, Parkinson's disease, Amyotrophic lateral sclerosis,Progressive supranuclear palsy, Pick's disease, several polyglutamineneurological disorders a such as Huntington, Spinocerebullar ataxia typeI, Spinal and bulbar muscular atrophy, Dentatorubal pallidoluysian, andMyotonic dystrophy, as well as Spongiform encephalopathies, such asHereditary Creutzfeldt-Jakob disease, Fabry disease,Straussler-Scheinker syndrome, COPD, dry-eye disease, and Sjogren'sdisease.

In one embodiment, the second therapeutic agent is an agent useful inthe treatment of cystic fibrosis.

In one embodiment, the second therapeutic agent is an agent useful inthe treatment of COPD.

In one embodiment, the second therapeutic agent is an agent useful inthe treatment of Parkinson's disease.

In one embodiment, the second therapeutic agent is an agent useful inthe treatment of a bile duct disorder or a kidney ion channel disorder,including, but not limited to, Bartter's syndrome and Dent's disease.

In one embodiment, the second therapeutic agent is VX-809 (lumacaftor)or VX-661.

In another embodiment, the invention provides separate dosage forms of acompound of this invention and one or more of any of the above-describedsecond therapeutic agents, wherein the compound and second therapeuticagent are associated with one another. The term “associated with oneanother” as used herein means that the separate dosage forms arepackaged together or otherwise attached to one another such that it isreadily apparent that the separate dosage forms are intended to be soldand administered together (within less than 24 hours of one another,consecutively or simultaneously).

In the pharmaceutical compositions of the invention, the compound of thepresent invention is present in an effective amount. As used herein, theterm “effective amount” refers to an amount which, when administered ina proper dosing regimen, is sufficient to treat the target disorder.

The interrelationship of dosages for animals and humans (based onmilligrams per meter squared of body surface) is described in Freireichet al., Cancer Chemother. Rep, 1966, 50: 219. Body surface area may beapproximately determined from height and weight of the subject. See,e.g., Scientific Tables, Geigy Pharmaceuticals, Ardsley, N.Y., 1970,537.

In one embodiment, an effective amount of a compound of this inventioncan range from about 0.02 to 2500 mg per treatment. In more specificembodiments the range is from about 0.2 to 1250 mg or from about 0.4 to500 mg or most specifically from 2 to 250 mg per treatment. Treatmenttypically is administered one to two times daily. In one embodiment, thecompound of the invention is administered two times daily in an amountbetween 50 and 300 mg each time. In one embodiment, the compound of theinvention is administered once daily in an amount between 100 to 500 mg.In the foregoing embodiments, the compound is administered optionally incombination with a second agent. Examples of second agents include CFTRcorrectors, such as lumacaftor or VX-661. In some embodiments whereinthe compound is administered optionally in combination with a secondagent, the amount of compound is administered twice daily at between 100mg and 300 mg each time, such as between 150 mg and 250 mg each time. Inother embodiments wherein the compound is administered optionally incombination with a second agent, the amount of compound is administeredthree times daily at between 100 mg and 300 mg each time, such asbetween 150 mg and 250 mg each time.

Effective doses will also vary, as recognized by those skilled in theart, depending on the diseases treated, the severity of the disease, theroute of administration, the sex, age and general health condition ofthe subject, excipient usage, the possibility of co-usage with othertherapeutic treatments such as use of other agents and the judgment ofthe treating physician.

For pharmaceutical compositions that comprise a second therapeuticagent, an effective amount of the second therapeutic agent is betweenabout 20% and 100% of the dosage normally utilized in a monotherapyregime using just that agent. Preferably, an effective amount is betweenabout 70% and 100% of the normal monotherapeutic dose. The normalmonotherapeutic dosages of these second therapeutic agents are wellknown in the art. See, e.g., Wells et al., eds., PharmacotherapyHandbook, 2nd Edition, Appleton and Lange, Stamford, Conn. (2000); PDRPharmacopoeia, Tarascon Pocket Pharmacopoeia 2000, Deluxe Edition,Tarascon Publishing, Loma Linda, Calif. (2000), each of which referencesare incorporated herein by reference in their entirety.

It is expected that some of the second therapeutic agents referencedabove will act synergistically with the compounds of this invention.When this occurs, it will allow the effective dosage of the secondtherapeutic agent and/or the compound of this invention to be reducedfrom that required in a monotherapy. This has the advantage ofminimizing toxic side effects of either the second therapeutic agent ofa compound of this invention, synergistic improvements in efficacy,improved ease of administration or use and/or reduced overall expense ofcompound preparation or formulation.

Methods of Treatment

In another embodiment, the invention provides a method of potentiatingthe activity of CFTR in an infected cell, comprising contacting such acell with a compound of Formula I herein, or a pharmaceuticallyacceptable salt thereof.

According to another embodiment, the invention provides a method oftreating a disease that is beneficially treated by VX-770 in a subjectin need thereof, comprising the step of administering to the subject aneffective amount of a compound or a composition of this invention. Inone embodiment the subject is a patient in need of such treatment. Suchdiseases include cystic fibrosis, Hereditary emphysema, Hereditaryhemochromatosis, Coagulation-Fibrinolysis deficiencies, such as ProteinC deficiency, Type 1 hereditary angioedema, Lipid processingdeficiencies, such as Familial hypercholesterolemia, Type 1chylomicronemia, Abetalipoproteinemia, Lysosomal storage diseases, suchas I-cell disease/Pseudo-Hurler, Mucopolysaccharidoses,Sandhof/Tay-Sachs, Crigler-Najjar type II,Polyendocrinopathy/Hyperinsulemia, Diabetes mellitus, Laron dwarfism,Myleoperoxidase deficiency, Primary hypoparathyroidism, Melanoma,Glycanosis CDG type 1, Hereditary emphysema, Congenital hyperthyroidism,Osteogenesis imperfecta, Hereditary hypofibrinogenemia, ACT deficiency,Diabetes insipidus (DI), Neurophyseal DI, Neprogenic DI, Charcot-MarieTooth syndrome, Perlizaeus-Merzbacher disease, neurodegenerativediseases such as Alzheimer's disease, Parkinson's disease, Amyotrophiclateral sclerosis, Progressive supranuclear palsy, Pick's disease,several polyglutamine neurological disorders such as Huntington,Spinocerebullar ataxia type I, Spinal and bulbar muscular atrophy,Dentatorubal pallidoluysian, and Myotonic dystrophy, as well asSpongiform encephalopathies, such as Hereditary Creutzfeldt-Jakobdisease, Fabry disease, Straussler-Scheinker syndrome, COPD, dry-eyedisease, and Sjogren's disease.

In one embodiment, a compound of this invention is used to treat cysticfibrosis in a subject such as a patient in need thereof. In oneembodiment, a compound of this invention is used to treat COPD in asubject such as a patient in need thereof. In an example of either ofthe foregoing embodiments, the compound is administered by nasal aerosolor inhalation. In another example of either of the foregoingembodiments, the compound is administered orally.

In one embodiment, a compound of this invention is used to treatParkinson's Disease in a subject such as a patient in need thereof.

In one embodiment, a compound of this invention is used to treat a bileduct disorder or a kidney ion channel disorder, including, but notlimited to, Bartter's syndrome and Dent's disease in a subject such as apatient in need thereof.

In another embodiment, any of the above methods of treatment comprisesthe further step of co-administering to the subject in need thereof oneor more second therapeutic agents. The choice of second therapeuticagent may be made from any second therapeutic agent known to be usefulfor co-administration with VX-770. The choice of second therapeuticagent is also dependent upon the particular disease or condition to betreated. Examples of second therapeutic agents that may be employed inthe methods of this invention are those set forth above for use incombination compositions comprising a compound of this invention and asecond therapeutic agent.

In particular, the combination therapies of this invention includeco-administering a compound of Formula I or a pharmaceuticallyacceptable salt thereof and a second therapeutic agent such as VX-809(lumacaftor) or VX-661, to a subject in need thereof for treatment.

The term “co-administered” as used herein means that the secondtherapeutic agent may be administered together with a compound of thisinvention as part of a single dosage form (such as a composition of thisinvention comprising a compound of the invention and an secondtherapeutic agent as described above) or as separate, multiple dosageforms. Alternatively, the additional agent may be administered prior to,consecutively with, or following the administration of a compound ofthis invention. In such combination therapy treatment, both thecompounds of this invention and the second therapeutic agent(s) areadministered by conventional methods. The administration of acomposition of this invention, comprising both a compound of theinvention and a second therapeutic agent, to a subject does not precludethe separate administration of that same therapeutic agent, any othersecond therapeutic agent or any compound of this invention to saidsubject at another time during a course of treatment.

Effective amounts of these second therapeutic agents are well known tothose skilled in the art and guidance for dosing may be found in patentsand published patent applications referenced herein, as well as in Wellset al., eds., Pharmacotherapy Handbook, 2nd Edition, Appleton and Lange,Stamford, Conn. (2000); PDR Pharmacopoeia, Tarascon Pocket Pharmacopoeia2000, Deluxe Edition, Tarascon Publishing, Loma Linda, Calif. (2000),and other medical texts. However, it is well within the skilledartisan's purview to determine the second therapeutic agent's optimaleffective-amount range.

In one embodiment of the invention, where a second therapeutic agent isadministered to a subject, the effective amount of the compound of thisinvention is less than its effective amount would be where the secondtherapeutic agent is not administered. In another embodiment, theeffective amount of the second therapeutic agent is less than itseffective amount would be where the compound of this invention is notadministered. In this way, undesired side effects associated with highdoses of either agent may be minimized. Other potential advantages(including without limitation improved dosing regimens and/or reduceddrug cost) will be apparent to those of skill in the art.

In yet another aspect, the invention provides the use of a compound ofFormula I, or a pharmaceutically acceptable salt thereof, alone ortogether with one or more of the above-described second therapeuticagents in the manufacture of a medicament, either as a singlecomposition or as separate dosage forms, for treatment or prevention ina subject of a disease, disorder or symptom set forth above. Anotheraspect of the invention is a compound of Formula I, or apharmaceutically acceptable salt thereof, for use in the treatment orprevention in a subject of a disease, disorder or symptom thereofdelineated herein.

EXAMPLES Example 1. Synthesis ofN-(2,4-Di-(tert-butyl-d₉)-3,6-d₂-5-hydroxyphenyl)-4-oxo-1,4-dihydroquinoline-3-carboxamide(Compound 110)

Compound 110 was prepared as outlined in Scheme 5 below.

Step 1. 2,4-Di-(tert-butyl-d₉)-3,5,6-d₃-phenol (4a)

Intermediate 4a was prepared according to the procedure described forthe synthesis for 2,4-di-tert-butyl-3,5-d₂-phenol employing tert-butylchloride-d₉ in place of tert-butylchloride (Kurahashi, T.; Hada, M.;Fujii, H. J. Am. Chem. Soc. 2009, 131, 12394-12405): To a solution ofphenol-d₆ (459 mg, 4.59 mmol, 99 atom % D, Sigma Aldrich) and tert-butylchloride-d₉ (2.50 mL, 23.0 mmol, 98 atom % D, Cambridge IsotopeLaboratories, Inc.) in 1,2-dichloroethane (10.0 mL) was added ReBr(CO)₅(19.0 mg, 0.0459 mmol). The reaction mixture was stirred at 85° C. for15 hours at which time additional tert-butyl chloride-d₉ (2.50 mL, 23.0mmol, 98 atom % D, Cambridge Isotope Laboratories, Inc.) and ReBr(CO)₅(19.0 mg, 0.0459 mmol) was added. Stirring was continued at 85° C. for 2hours, the mixture was cooled to room temperature, concentrated in vacuoand purified by column chromatography (SiO₂, 30% CH₂Cl₂/heptanes) toafford 4a (0.789 g, 76% yield) as a light yellow oil. MS (ESI) 228.1[(M+H)⁺].

Step 2. 2,4-Di-(tert-butyl-d₉)-3,5,6-d₃-phenyl methyl carbonate (20)

To a solution of 4a (2.72 g, 12.0 mmol), triethylamine (3.33 mL, 23.9mmol) and N,N-dimethylaminopyridine (73.0 mg, 0.598 mmol) in CH₂Cl₂(30.0 mL) at 0° C. was added methyl chloroformate (1.38 mL, 17.9 mmol).The reaction mixture was stirred at room temperature for 15 hours thenwas diluted with 10% ethyl acetate/heptanes and filtered through asilica plug. The silica plug was then rinsed with additional 10% ethylacetate/heptanes. The filtrate was combined, and concentrated in vacuoto provide 20 (2.40 g, 70% yield) as a light yellow oil which wascarried forward without purification.

Step 3. 2,4-Di-(tert-butyl-d₉)-3,6-d₂-5-nitrophenol (21)

To a solution of 20 (2.40 g, 8.41 mmol) in sulfuric acid (1.00 mL) at 0°C. was added a 1:1 mixture of sulfuric acid and nitric acid (2.00 mL)dropwise. The reaction mixture was then stirred at room temperature for2 hours then slowly added to ice water with vigorous stirring. Theresulting slurry was extracted with ethyl acetate (3×100 mL) and thecombined organic layers were dried (Na₂SO₄), filtered and concentratedto afford an amber oil containing a mixture of regioisomers. This crudeoil was then taken up in MeOH (50 mL) and KOH (1.54 g, 27.5 mmol) wasadded. The reaction mixture was stirred at room temperature for 2 hoursthen was acidified to pH=2 with concentrated HCl. The resulting solutionwas extracted with diethyl ether (3×100 mL), dried (MgSO₄), filtered andconcentrated. The residue was then purified via column chromatography(SiO₂, 0-5% ethyl acetate/heptanes) to afford 21 (526 mg, 23%) as alight yellow solid. MS (ESI) 270.3 [(M−H)⁻].

Step 4. 5-Amino-2,4-di-(tert-butyl-d₉)-3,6-d₂-phenol (22)

A solution of 21 (526 mg, 1.94 mmol) and ammonium formate (489 mg, 7.75mmol) in ethanol (25.0 mL) was heated to the point of reflux. At thistime, 10% Pd/C (250 mg, 50% wet) was added in small portions and thereaction mixture was stirred at reflux for 2 hours. The mixture was thencooled to room temperature, diluted with THF, filtered through Celite®and concentrated in vacuo to afford 22 (473 mg, 100%) as a tan solid. MS(ESI) 242.4 [(M+H)⁺].

Step 5.N-(2,4-Di-(tert-butyl-d₉)-3,6-d₂-5-hydroxyphenyl)-4-oxo-1,4-dihydroquinoline-3-carboxamide(Compound 110)

To a solution of 22 (250 mg, 1.04 mmol),4-oxo-1,4-dihydroquinoline-3-carboxylic acid (23, purchased from MatrixScientific, 98.0 mg, 0.518 mmol) and N,N-diisopropylethylamine (181 μL,1.04 mmol) in DMF (5.00 mL) was added HATU (197 mg, 0.518 mmol). Thereaction mixture was stirred at room temperature for 3 hours then wasdiluted with saturated NaHCO₃ and extracted with ethyl acetate (3×50mL). The combined organic extracts were washed with water (3×20 mL),dried (Na₂SO₄), filtered and concentrated in vacuo. The resultingresidue was purified via column chromatography (SiO₂, 0-70% ethylacetate/heptanes) to afford Compound 110 (77.0 mg, 36% Yield) as a whitesolid. ¹H NMR (d₆-DMSO, 400 MHz): δ 12.87 (br s, 1H), 11.80 (s, 1H),9.18 (s, 1H), 8.86 (s, 1H), 8.32 (d, J=8.2 Hz, 1H), 7.81 (t, J=7.9 Hz,1H), 7.76 (t, J=8.2 Hz, 1H), 7.51 (t, J=7.4 Hz, 1H), 7.10 (s, 0.2H)*; MS(ESI) 413.5 [(M+H)⁺]. *The ¹H NMR signal at 7.10 ppm indicatesapproximately 80% deuterium incorporation at one of the two deuteratedaryl positions. The absence of signals at 7.20 ppm and 1.37 ppm indicatehigh levels of incorporation (>95%) at the remaining deuteratedpositions.

Example 2. Synthesis ofN-(2-(tert-Butyl)-4-(tert-butyl-d₉)-6-d-5-hydroxyphenyl)-4-oxo-1,4-dihydroquinoline-3-carboxamide(Compound 125)

Compound 125 was prepared as outlined in Scheme 6 below.

Step 1. 2-(tert-Butyl-d₉)-4-(tert-butyl)-6-d-phenol (7)

To a solution of 4-tert-butyl phenol (3.43 g, 22.7 mmol) and tert-butylalcohol-d10 (3.00 mL, 31.8 mmol, 98 atom % D, Cambridge IsotopeLaboratories, Inc.) in dichloromethane (40.0 mL) was added D₂SO₄ (1.50mL, 99.5 atom % D, Sigma-Aldrich). The reaction was stirred at roomtemperature for 15 hours then was diluted with water and extracted withdichloromethane (3×100 mL). The organic layers were combined, washedwith saturated NaHCO₃, dried (Na₂SO₄), filtered and concentrated invacuo. The resulting oil was purified by column chromatography (SiO₂,0-15% ethyl acetate/heptanes) to afford 7 (4.04 g, 83% yield) as a clearoil. ¹H NMR (d₆-DMSO, 400 MHz) δ 9.04 (s, 1H), 7.12 (d, J=2.4 Hz, 1H),6.98 (dd, J=3.8, 2.5 Hz, 1H), 6.67 (d, J=8.3 Hz, 0.3H), 1.22 (s, 10H).

Step 2. 2-(tert-Butyl-d₉)-4-(tert-butyl)-6-d-phenyl methyl carbonate (8)

To a solution of 7 (4.04 g, 18.8 mmol), triethylamine (5.24 mL, 37.6mmol) and N,N-dimethylaminopyridine (115 mg, 0.940 mmol) in CH₂Cl₂ (40.0mL) at 0° C. was added methyl chloroformate (2.17 mL, 28.2 mmol). Thereaction was stirred at room temperature for 15 hours and additionaltriethylamine (1.30 mL, 9.33 mmol) and methyl chloroformate (0.550 mL,7.15 mmol) were added. After stirring for an additional 1 hour thereaction was diluted with 10% ethyl acetate/heptanes and filteredthrough a silica plug. The silica plug was then rinsed with additional10% ethyl acetate/heptanes. The filtrate was combined and concentratedin vacuo to provide 8 (4.69 g, 91% yield) as a light yellow oil whichwas carried forward without purification. ¹H NMR (d₆-DMSO, 400 MHz) δ7.33 (d, J=2.4 Hz, 1H), 7.30-7.20 (m, 1H), 7.06 (d, J=8.5 Hz, 0.3H),3.84 (d, J=0.7 Hz, 3H), 1.28 (s, 9H).

Step 3. 2-(tert-Butyl-d₉)-4-(tert-butyl)-6-d-5-nitro-phenol (9)

To a solution of 8 (4.69 g, 17.2 mmol) in sulfuric acid (2.00 mL) at 0°C. was added a 1:1 mixture of sulfuric acid and nitric acid (4.00 mL)dropwise. The reaction was then stirred at room temperature for twohours then slowly added to ice water with vigorous stirring. Theresulting slurry was extracted with ethyl acetate (3×100 mL) and thecombined organic layers were dried (Na₂SO₄), filtered and concentratedto afford an amber oil containing a mixture of regio-isomers. This crudeoil was then taken up in MeOH (100 mL) and KOH (3.50 g) was added. Thereaction stirred at room temperature for 2 hours then was acidified topH=2 with concentrated HCl. The resulting solution was extracted withdiethyl ether (3×100 mL), dried (MgSO₄), filtered and concentrated. Theresidue was then purified via column chromatography (SiO₂, 0-5% ethylacetate/heptanes) to afford 9 (1.33 g, 30%) as a light yellow solid. MS(ESI) 260.2 [(M−H)⁻].

Step 4. 5-Amino-2-(tert-butyl-d₉)-4-(tert-butyl)-6-d-phenol (10)

A solution of 9 (1.33 g, 5.11 mmol) and ammonium formate (1.29 g, 20.4mmol) in ethanol (60.0 mL) was heated to reflux. At this time, 10% Pd/C(650 mg, 50% wet) was added in small portions and the reaction continuedto stir at reflux for two hours. The reaction was then cooled to roomtemperature, diluted with THF, filtered through Celite® and concentratedin vacuo to afford 10 (1.19 g, 100%) as a pink solid. MS (ESI) 232.3[(M+H)⁺].

Step 5.N-(2-(tert-Butyl)-4-(tert-butyl-d₉)-6-d-5-hydroxyphenyl)-4-oxo-1,4-dihydroquinoline-3-carboxamide(125)

To a solution of 10 (892 mg, 3.87 mmol),4-oxo-1,4-dihydroquinoline-3-carboxylic acid (11, purchased from MatrixScientific, 366 mg, 1.93 mmol) and N,N-diisopropylethylamine (674 μL,3.87 mmol) in DMF (20.0 mL) was added HATU (734 mg, 1.93 mmol). Thereaction was stirred at room temperature for three hours then wasdiluted with saturated NaHCO₃ and extracted with ethyl acetate (3×50mL). The combined organic extracts were washed with water (3×20 mL),dried (Na₂SO₄), filtered and concentrated in vacuo. The resultingresidue was purified via column chromatography (SiO₂, 0-70% ethylacetate/heptanes) to afford 125 (277 mg, 36% Yield) as a white solid. ¹HNMR (d₆-DMSO, 400 MHz) δ 12.88 (s, 1H), 11.81 (s, 1H), 9.19 (s, 1H),8.86 (s, 1H), 8.32 (dd, J=8.1, 1.4 Hz, 1H), 7.86-7.77 (m, 1H), 7.75 (d,J=8.2 Hz, 1H), 7.51 (s, 1H), 7.15 (s, 1H), 7.09 (s, 0.3H)*, 1.37 (s,9H); MS (ESI) 403.3 [(M+H)⁺]. *The 1H NMR signal at 7.09 ppm indicatesapproximately 70% deuterium incorporation at one of the two arylpositions.

Example 3. Synthesis ofN-(2-(tert-Butyl)-4-(tert-butyl-d₉)-5-hydroxyphenyl)-4-oxo-1,4-dihydroquinoline-3-carboxamide(Compound 106)

Compound 106 was prepared as outlined in Scheme 7 below.

Step 1. 5-Amino-2-(tert-butyl-d₉)-4-(tert-butyl)-phenol (12)

Compound 10 (298 mg, 1.29 mmol), prepared as disclosed in Example 2, wasdissolved in 5M HCl in 2-propanol (20 mL) and the reaction was stirredat room temperature for 15 hours. The reaction was then concentrated invacuo and taken back up in 5M HCl in 2-propanol (20 mL). After stirringfor an additional 15 hours at room temperature, the reaction wasconcentrated in vacuo and diluted with saturated aqueous sodiumbicarbonate (100 mL). The resulting aqueous solution was extracted withdichloromethane (3×50 mL). The organic layers were combined, dried(Na₂SO₄), filtered and concentrated in vacuo to afford 12 (240 mg, 81%)as a pink solid. ¹H NMR (d6-DMSO, 400 MHz) δ 8.62 (s, 1H), 6.83 (s, 1H),6.08 (s, 1H), 1.27 (s, 9H).

Step 2.N-(2-(tert-Butyl)-4-(tert-butyl-d₉)-5-hydroxyphenyl)-4-oxo-1,4-dihydroquinoline-3-carboxamide(106)

To a solution of 12 (240 mg, 1.04 mmol),4-oxo-1,4-dihydroquinoline-3-carboxylic acid (11, purchased from MatrixScientific, 99 mg, 0.521 mmol) and N,N-diisopropylethylamine (181 μL,1.04 mmol) in DMF (6.00 mL) was added HATU (198 mg, 0.521 mmol). Thereaction was stirred at room temperature for three hours then wasdiluted with saturated NaHCO₃ and extracted with ethyl acetate (3×50mL). The combined organic extracts were washed with water (3×20 mL),dried (Na₂SO₄), filtered and concentrated in vacuo. The resultingresidue was purified via column chromatography (SiO₂, 0-70% ethylacetate/heptanes) to afford 106 (80 mg, 38% Yield) as a white solid. ¹HNMR (d₆-DMSO, 400 MHz) δ 12.88 (s, 1H), 11.81 (s, 1H), 9.19 (s, 1H),8.86 (s, 1H), 8.32 (dd, J=8.1, 1.4 Hz, 1H), 7.86-7.77 (m, 1H), 7.75 (d,J=8.2 Hz, 1H), 7.51 (s, 1H), 7.15 (s, 1H), 7.09 (s, 1H), 1.37 (s, 9H);MS (ESI) 402.3 [(M+H)⁺].

In one batch run, the isotopic enrichment factor for X⁷ in 106 was foundto be about 466.7 (7% deuterium incorporation). In one batch run, theisotopic enrichment factor for X⁷ in 106 was found to be about 733.3(11% deuterium incorporation).

Example 4. Synthesis ofN-(2,4-Di-(tert-butyl-d₉)-5-hydroxyphenyl)-4-oxo-1,4-dihydroquinoline-3-carboxamide(Compound 105)

Compound 105 was prepared as outlined in Scheme 5 below.

Step 1. 2,4,6-d₃-Phenol-OD (32)

Phenol (20.0 g, 212 mmol) was added to a 3.5M solution of DCl in D₂O(200 mL) in a sealed tube. The mixture then stirred at 140° C. for 72hours then was cooled to room temperature and extracted with CH₂Cl₂(3×100 mL). The combined organic layers were dried (Na₂SO₄), filteredand concentrated to afford a light pink solid (19.2 g, 93% yield). ¹HNMR (d₆-DMSO, 400 MHz) δ 9.34 (s, 0.22H, OH), 7.15 (s, 2H), 6.76 (m,0.14H). (The peak at 6.76 ppm represents the hydrogen atoms at the 2, 4and 6 positions, therefore an integration of 0.14 indicates the materialobtained has ˜95% deuterium incorporation at these positions.)

Step 2. 2-d-4,6-bis(1,1,1,3,3,3-d₆-2-(methyl-d₃)propan-2-yl)phenol (33)

To a solution of 32 (2.08 g, 21.2 mmol) and tert-butyl alcohol-d10 (5.00mL, 53.0 mmol, 98 atom % D, Cambridge Isotope Laboratories, Inc.) indichloromethane (40.0 mL) was added D₂SO₄ (1.71 mL, 99.5 atom % D,Sigma-Aldrich). The reaction stirred at room temperature for 15 hoursthen was diluted with water and extracted with dichloromethane (3×100mL). The organic layers were combined, washed with saturated NaHCO₃,dried (Na₂SO₄), filtered and concentrated in vacuo. The resulting oilwas purified by column chromatography (SiO₂, 0-15% ethylacetate/heptanes) to afford 33 (1.45 g, 30% yield) as a clear oil. ¹HNMR (d₆-DMSO, 400 MHz) δ 9.03 (s, 1H), 7.11 (d, J=2.5 Hz, 1H), 6.98 (td,J=4.1, 2.5 Hz, 1H), 6.67 (d, J=8.3 Hz, 0.5H), 1.28 (s, 0.18H), 1.17 (s,0.21H). (The peak at 6.67 ppm integrating for 0.5H indicates isotopicerosion to approximately 50% D at the 2-position during the course ofthe reaction. The peaks at 1.28 and 1.17 ppm represent the hydrogencontent of the t-butyl groups, therefore the integrations of 0.18 and0.21 indicate approximately 98% D incorporation for both.)

Step 3. Methyl(2-d-4,6-bis(1,1,1,3,3,3-d₆-2-(methyl-d₃)-propan-2-yl)phenyl) carbonate(34)

To a solution of 33 (1.45 g, 6.43 mmol), triethylamine (2.24 mL, 16.1mmol) and N,N-dimethylaminopyridine (40.0 mg, 0.322 mmol) in CH₂Cl₂(15.0 mL) at 0° C. was added methyl chloroformate (0.990 mL, 12.9 mmol).The reaction stirred at room temperature for 15 hours then was dilutedwith 10% ethyl acetate/heptanes and filtered through a silica plug. Thesilica plug was then rinsed with additional 10% ethyl acetate/heptanes.The filtrate was combined, and concentrated in vacuo to provide 34 (1.78g, 98% yield) as a light yellow oil which was carried forward withoutpurification.

Step 4.2-d-4,6-bis(1,1,1,3,3,3-d₆-2-(methyl-d₃)propan-2-yl)-3-nitrophenol (35)

To a solution of 34 (1.78 g, 6.28 mmol) in sulfuric acid (1.00 mL) at 0°C. was added a 1:1 mixture of sulfuric acid and nitric acid (2.00 mL)dropwise. The reaction was then stirred at room temperature for twohours then slowly added to ice water with vigorous stirring. Theresulting slurry was extracted with ethyl acetate (3×100 mL) and thecombined organic layers were dried (Na₂SO₄), filtered and concentratedto afford an amber oil containing a mixture of regioisomers. This crudeoil was then taken up in MeOH (20 mL) and KOH (664 mg, 11.8 mmol) wasadded. The reaction stirred at room temperature for 2 hours then wasacidified to pH=2 with concentrated HCl. The resulting solution wasextracted with diethyl ether (3×100 mL), dried (MgSO₄), filtered andconcentrated. The residue was then purified via column chromatography(SiO₂, 0-5% ethyl acetate/heptanes) to afford 35 (319 mg, 19%) as alight yellow solid. MS (ESI) 269.3 [(M−H)⁻].

Step 5.3-Amino-2-d-4,6-bis(1,1,1,3,3,3-d₆-2-(methyl-d₃)propan-2-yl)phenol (36)

A solution of 35 (319 mg, 1.18 mmol) and ammonium formate (298 mg, 4.72mmol) in ethanol (20.0 mL) was heated to reflux. At this time, 10% Pd/C(160 mg, 50% wet) was added in small portions and the reaction continuedto stir at reflux for two hours. The reaction was then cooled to roomtemperature, diluted with THF and filtered through Celite® andconcentrated in vacuo to afford 36 (279 mg, 98%) as a tan solid. MS(ESI) 241.3 [(M+H)⁺].

Step 6. 3-Amino-4,6-bis(1,1,1,3,3,3-d₆-2-(methyl-d₃)propan-2-yl)phenol(37)

Compound 36 (279 mg, 1.16 mmol) was dissolved in 5M HCl in 2-propanol(20 mL) and the reaction stirred at room temperature for 15 hours. Thereaction was then concentrated in vacuo and taken back up in 5M HCl in2-propanol (20 mL). After stirring for an additional 15 hours at roomtemperature, the reaction was concentrated in vacuo and diluted withsaturated aqueous sodium bicarbonate (100 mL). The resulting aqueoussolution was extracted with dichloromethane (3×50 mL). The organiclayers were combined, dried (Na₂SO₄), filtered and concentrated in vacuoto afford 37 (255 mg, 91%) as a pink solid. MS (ESI) 240.3 [(M+H)⁺].

Step 7.N-(2,4-Di-(tert-butyl-d₉)-5-hydroxyphenyl)-4-oxo-1,4-dihydroquinoline-3-carboxamide(105)

To a solution of 37 (255 mg, 1.06 mmol),4-oxo-1,4-dihydroquinoline-3-carboxylic acid (purchased from MatrixScientific, 100 mg, 0.532 mmol) and N,N-diisopropylethylamine (185 μL,1.06 mmol) in DMF (6.00 mL) was added HATU (202 mg, 0.532 mmol). Thereaction stirred at room temperature for three hours then was dilutedwith saturated NaHCO₃ and extracted with ethyl acetate (3×50 mL). Thecombined organic extracts were washed with water (3×20 mL), dried(Na₂SO₄), filtered and concentrated in vacuo. The resulting residue waspurified via column chromatography (SiO₂, 0-70% ethyl acetate/heptanes)to afford 105 (92 mg, 42% yield) as a white solid. ¹H NMR (d₆-DMSO, 400MHz) δ 12.88 (s, 1H), 11.81 (s, 1H), 9.19 (s, 1H), 8.86 (s, 1H), 8.32(dd, J=8.1, 1.5 Hz, 1H), 7.86-7.69 (m, 2H), 7.51 (ddd, J=8.2, 6.7, 1.4Hz, 1H), 7.14 (s, 1H), 7.10 (s, 1H), 1.32 (s, 0.2H), 1.30 (s, 0.18H).(The peaks at 1.32 and 1.30 ppm represent the hydrogen content of thet-butyl groups, therefore the integrations of 0.20 and 0.18 indicateapproximately 98% D incorporation for both.) MS (ESI) 411.4 [(M+H)⁺].

In one batch run, the isotopic enrichment factor for X⁷ in 105 was foundto be about 266.7 (4% deuterium incorporation). In one batch run, theisotopic enrichment factor for X⁷ in 105 was found to be about 333.3 (5%deuterium incorporation).

Example 5. Synthesis ofN-(2-(tert-Butyl-d₉)-4-(tert-butyl)-3-d-5-hydroxyphenol)-4-oxo-1,4-dihydroquinoline-3-carboxamide(Compound 123)

Compound 123 was prepared as outlined in Scheme 6 below.

Step 1. 4-tert-butylphenol-d₁₃ (42)

To a solution of phenol-d6 (2.06 g, 20.6 mmol, 99 atom % D,Sigma-Aldrich) and tert-butyl chloride-d9 (6.73 mL, 61.8 mmol, 98 atom %D, Cambridge Isotope Laboratories, Inc.) in 1,2-dichloroethane (40.0 mL)was added ReBr(CO)₅ (84.0 mg, 0.210 mmol). The reaction stirred at 80°C. for 15 hours at which time the reaction was cooled to roomtemperature, concentrated in vacuo and purified by column chromatography(SiO₂, 0-10% EtOAc/heptanes) to afford 42 (1.95 g, 58% yield) as acolorless crystalline solid. MS (ESI) 162.1[(M−H)⁻].

Step 2. 2-(tert-Butyl)-3,5,6-d₃-4-(tert-butyl-d₉)-phenol (43)

To a solution of 42 (1.95 g, 12.0 mmol) and tert-butyl alcohol-d10 (1.60mL, 16.7 mmol, 98 atom % D, Cambridge Isotope Laboratories, Inc.) indichloromethane (30 mL) was added D₂SO₄ (0.900 mL, 99.5 atom % D,Sigma-Aldrich). The reaction stirred at room temperature for 15 hoursthen was diluted with water and extracted with dichloromethane (3×100mL). The organic layers were combined, washed with saturated NaHCO₃,dried (Na₂SO₄), filtered and concentrated in vacuo. The resulting oilwas purified by column chromatography (SiO₂, 0-15% ethylacetate/heptanes) to afford 43 (782 mg, 30% yield) as a clear oil.

Step 3. 2-(tert-Butyl)-3,5,6-d₃-4-(tert-butyl-d₉)phenyl methyl carbonate(44)

To a solution of 43 (782 mg, 3.59 mmol), triethylamine (1.25 mL, 8.98mmol) and N,N-dimethylaminopyridine (22.0 mg, 0.180 mmol) in DCM (10.0mL) at 0° C. was added a solution of methyl chloroformate (0.552 mL,7.17 mmol) in DCM (2.00 mL) dropwise over 30 minutes. The reactionstirred at room temperature for 2 hours then was diluted with 20% ethylacetate/heptanes (50.0 mL) and filtered through a silica plug. Thesilica plug was then rinsed with additional 20% ethyl acetate/heptanes(3×50 mL). The filtrate was combined, and concentrated in vacuo toprovide 44 (930 mg, 94% yield) as a light yellow oil which was carriedforward without purification.

Step 4. 2-(tert-Butyl)-3,6-d₂-4-(tert-butyl-d₉)-5-nitrophenol (45)

To a solution of 44 (0.930 g, 3.36 mmol) in sulfuric acid (0.500 mL) at0° C. was added a 1:1 mixture of sulfuric acid and nitric acid (1.00 mL)dropwise. The reaction was then stirred at room temperature for twohours then slowly added to ice water with vigorous stirring. Theresulting slurry was extracted with diethyl ether (3×50 mL) and thecombined organic layers were dried (Na₂SO₄), filtered and concentrated.The resulting oil was purified via column chromatography (SiO₂, 0-10%EtOAc/heptanes) to afford the nitrophenyl methyl carbonate as a mixtureof 2 regioisomers. The mixture of regioisomers was then taken up in MeOH(5.00 mL) and KOH (205 mg, 3.66 mmol) was added. The reaction stirred atroom temperature for 2 hours then was diluted with 1N HCl. The resultingsolution was extracted with DCM (3×25 mL), dried (Na₂SO₄), filtered andconcentrated. The resulting orange solid was then slurried in hexane(25.0 mL) at 0° C. for 20 minutes. The solids were then collected byfiltration, washed with additional cold hexane, and dried to afford 45(52.0 mg, 19% yield) as a light yellow solid. MS (ESI) 260.2 [(M−H)⁻].

Step 5. 5-Amino-2-(tert-butyl)-3,6-d₂-4-(tert-butyl-d₉)-phenol (46)

A solution of 45 (52.0 mg, 0.199 mmol) and ammonium formate (50.0 mg,0.796 mmol) in ethanol (5.00 mL) was heated to reflux. At this time, 10%Pd/C (25.0 mg, 50% wet) was added in small portions and the reactioncontinued to stir at reflux for two hours. The reaction was then cooledto room temperature, diluted with THF and filtered through Celite® andconcentrated in vacuo to afford 46 (46.0 mg, 100%) as a tan solid. MS(ESI) 233.4 [(M+H)⁺].

Step 6. 5-Amino-2-(tert-butyl)-5-d-4-(tert-butyl-d₉)-phenol (47)

Compound 46 (46.0 mg, 0.198 mmol) was dissolved in 5M HCl in 2-propanol(10 mL) and the reaction stirred at room temperature for 15 hours. Thereaction was then concentrated in vacuo and re-dissolved in 5M HCl in2-propanol (10 mL). After stirring for an additional 15 hours at roomtemperature, the reaction was concentrated in vacuo and diluted withsaturated aqueous sodium bicarbonate (50 mL). The resulting aqueoussolution was extracted with dichloromethane (3×50 mL). The organiclayers were combined, dried (Na₂SO₄), filtered and concentrated in vacuoto afford 47 (42.0 mg, 91%) as a pink solid. MS (ESI) 232.3 [(M+H)⁺].

Step 7.N-(2-(tert-Butyl-d₉)-4-(tert-butyl)-3-d-5-hydroxyphenyl)-4-oxo-1,4-dihydroquinoline-3-carboxamide(123)

To a solution of 47 (40.0 mg, 0.173 mmol),4-oxo-1,4-dihydroquinoline-3-carboxylic acid (purchased from MatrixScientific, 16.0 mg, 0.0870 mmol) and N,N-diisopropylethylamine (30.0μL, 0.173 mmol) in DMF (2.00 mL) was added HATU (33.0 mg, 0.0870 mmol).The reaction stirred at room temperature for three hours then wasdiluted with saturated NaHCO₃ and extracted with ethyl acetate (3×50mL). The combined organic extracts were washed with water (3×20 mL),dried (Na₂SO₄), filtered and concentrated in vacuo. The resultingresidue was purified via column chromatography (SiO₂, 0-70% ethylacetate/heptanes) and dried in a vacuum oven at 50° C. to afford 123(11.0 mg, 31% yield) as a white solid. ¹H NMR (d₆-DMSO, 400 MHz) δ 12.88(s, 1H), 11.81 (s, 1H), 9.20 (s, 1H), 8.86 (s, 1H), 8.32 (dd, J=8.2, 1.4Hz, 1H), 7.86-7.71 (m, 2H), 7.51 (ddd, J=8.2, 6.8, 1.3 Hz, 1H), 7.10 (s,1H), 1.32 (s, 0.2H), 1.36 (s, 9.18H). (The peak at 1.36 represents thecombined hydrogen content of the t-butyl groups. Therefore, if we assumethe ortho t-butyl group is 100% H, the total integration of 9.18indicates the para t-butyl is integrating 0.18, which corresponds toapproximately 2% H or approximately 98% D.) MS (ESI) 403.3 [(M+H)⁺].

Example 6a. Evaluation of Metabolic Stability of Compound 110—HumanCYP3A4 Supersomes™

SUPERSOMES™ Assay.

7.5 mM stock solutions of test compounds, Compound 110 and ivacaftor,were prepared in DMSO. The 7.5 mM stock solutions were diluted to 50 mMin acetonitrile (ACN). Human CYP3A4 Supersomes™ (1000 pmol/mL, purchasedfrom BD Gentest™ Products and Services) were diluted to 62.5 pmol/mL in0.1 M potassium phosphate buffer, pH 7.4, containing 3 mM MgCl₂. Thediluted supersomes were added to wells of a 96-well polypropylene platein triplicate. A 10 mL aliquot of the 50 mM test compound was added tothe supersomes and the mixture was pre-warmed for 10 minutes. Reactionswere initiated by addition of pre-warmed NADPH solution. The finalreaction volume was 0.5 mL and contained 50 pmol/mL CYP3A4 Supersomes™,1.0 mM test compound, and 2 mM NADPH in 0.1 M potassium phosphatebuffer, pH 7.4, and 3 mM MgCl₂. The reaction mixtures were incubated at37° C., and 50 mL aliquots were removed at 0, 5, 10, 20, and 30 minutesand added to 96-well plates which contained 50 mL of ice-cold ACN withinternal standard to stop the reactions. The plates were stored at 4° C.for 20 minutes after which 100 mL of water was added to the wells of theplate before centrifugation to pellet precipitated proteins.Supernatants were transferred to another 96-well plate and analyzed foramounts of parent remaining by LC-MS/MS using an Applied Bio-systems API4000 mass spectrometer.

Data Analysis:

The in vitro half-lives (t_(1/2) values) for test compounds werecalculated from the slopes of the linear regression of LN (% parentremaining) vs incubation time relationship:in vitro t _(1/2)=0.693/k, where k=−[slope of linear regression of %parent remaining (ln) vs incubation time]

FIG. 1 shows a plot of the percentage of parent compound remaining overtime for Compound 110 and for ivacaftor in human cytochromeP450-specific SUPERSOMES™. The t_(1/2) values, and the percentageincrease (% Δ) in average t_(1/2), are shown in Table 4 below.

TABLE 4 Results of In Vitro Human Cytochrome P450-Specific SUPERSOMES ™t_(1/2) (min) Exper- Exper- Exper- AVE ± SD Compound iment 1 iment 2iment 3 % Δ Ivacaftor 5.2 6.0 5.8 5.7 ± 0.4 Compound 110 9.9 7.9 8.7 8.8± 0.8 55%

Table 4 shows that Compound 110 has a 55% longer half life in the assaythan ivacaftor.

Example 6b. Evaluation of Metabolic Stability of Compounds 105 and106—Human CYP3A4 Supersomes™

SUPERSOMES™ Assay.

7.5 mM stock solutions of test compounds, Compounds 105, 106 andivacaftor, were prepared in DMSO. The 7.5 mM stock solutions werediluted to 50 mM in acetonitrile (ACN). Human CYP3A4 Supersomes™ (1000pmol/mL, purchased from BD Gentest™ Products and Services) were dilutedto 62.5 pmol/mL in 0.1 M potassium phosphate buffer, pH 7.4, containing3 mM MgCl₂. The diluted supersomes were added to wells of a 96-wellpolypropylene plate in triplicate. A 10 mL aliquot of the 50 mM testcompound was added to the supersomes and the mixture was pre-warmed for10 minutes. Reactions were initiated by addition of pre-warmed NADPHsolution. The final reaction volume was 0.5 mL and contained 50 pmol/mLCYP3A4 Supersomes™, 1.0 mM test compound, and 2 mM NADPH in 0.1 Mpotassium phosphate buffer, pH 7.4, and 3 mM MgCl₂. The reactionmixtures were incubated at 37° C., and 50 mL aliquots were removed at 0,5, 10, 20, and 30 minutes and added to 96-well plates which contained 50mL of ice-cold ACN with internal standard to stop the reactions. Theplates were stored at 4° C. for 20 minutes after which 100 mL of waterwas added to the wells of the plate before centrifugation to pelletprecipitated proteins. Supernatants were transferred to another 96-wellplate and analyzed for amounts of parent remaining by LC-MS/MS using anApplied Bio-systems API 4000 mass spectrometer.

Data Analysis:

The in vitro half-lives (t_(1/2) values) for test compounds werecalculated from the slopes of the linear regression of LN (% parentremaining) vs incubation time relationship:in vitro t _(1/2)=0.693/k, where k=−[slope of linear regression of %parent remaining (ln) vs incubation time]

The t_(1/2) values, and the percentage increase (% Δ) in averaget_(1/2), for Compounds 105, 106 and for ivacaftor in human cytochromeP450-specific SUPERSOMES™, are shown in Table 5 below.

TABLE 5 Results of In Vitro Human Cytochrome P450-Specific SUPERSOMES ™t_(1/2) (min) Exper- Exper- AVE ± SD Compound iment 1 iment 2 % ΔIvacaftor 5.5 4.9 5.2 ± 0.4 Compound 106 7.4 7.3 7.4 ± 0.1 41% Compound105 7.6 8.0 7.8 ± 0.3 49%

Table 5 shows that Compound 106 has a 41% longer half-life in the assaythan ivacaftor, and that Compound 105 has a 49% longer half-life thanivacaftor.

Example 7. Evaluation of Pharmacokinetics in Rats for Compounds 105 and106

Ivacaftor, compound 105 and compound 106 were discrete dosed to rats viaoral gavage (PO). Each compound was administered at a dose of 10 mg/kgto three rats (N=3 rats/compound; total of 9 rats in the study). Eachcompound was formulated in 100% PEG400 at a concentration of 2 mg/mL.Blood samples were collected from each rat at 15 and 30 minutes, and 1,2, 4, 6, 8, 12, 24, 48, and 72 hours post-dose. Blood samples werecentrifuged to obtain plasma. Plasma samples were analyzed forconcentrations of the dosed compound at each time point using LC-MS/MS.The limit of quantitation of each compound was 1 ng/mL.

The rat t_(1/2) values (determined by non-compartmental analysis usingWinNonlin software) for each compound are shown in Table 6 below:

TABLE 6 Compd Animal t_(1/2) Dosed ID (hr) Ivacaftor 1.1 9.03 1.2 12.431.3 10.01 Mean 10.49 SD 1.75 SE 1.01 CV % 16.70 106 2.1 13.50 2.2 14.282.3 11.92 Mean 13.24 (+26% ^(a)) SD 1.20 SE 0.69 CV % 9.10 105 3.1 13.803.2 16.33 3.3 14.39 Mean 14.84 (+42% ^(a)) SD 1.32 SE 0.76 CV % 8.90^(a) % change relative to ivacaftor t_(1/2) value

Table 6 shows that Compound 106 has a 26% longer mean half-life thanivacaftor, and that Compound 105 has a 42% longer mean half-life thanivacaftor.

Without further description, it is believed that one of ordinary skillin the art can, using the preceding description and the illustrativeexamples, make and utilize the compounds of the present invention andpractice the claimed methods. It should be understood that the foregoingdiscussion and examples merely present a detailed description of certainpreferred embodiments. It will be apparent to those of ordinary skill inthe art that various modifications and equivalents can be made withoutdeparting from the spirit and scope of the invention.

I claim:
 1. Compound 106

or a pharmaceutically acceptable salt thereof, obtained by a processcomprising reacting compound 12

with compound 6

to form a compound of Formula 106, wherein any atom not designated asdeuterium is present at its natural isotopic abundance, and wherein thepercentage of isotopic enrichment for each designated deuterium is atleast 90%, and optionally treating compound 106 with a base to produce apharmaceutically acceptable salt of Compound
 106. 2. The compound ofclaim 1, wherein the reaction of compound 12 with compound 6 isperformed in the presence of a coupling agent and base.
 3. The compoundof claim 2, wherein the coupling agent is(N,N,N′,N′)-tetramethyl-O-(7-azabenzotriazol-1-yl)uroniumhexafluorophosphate and the base is N,N′-diisopropylethylamine.
 4. Thecompound of claim 1, wherein compound 12 is produced by convertingcompound 10

into compound 12


5. The compound of claim 4, wherein compound 10 is converted to compound12 with a reducing agent.
 6. The compound of claim 5, wherein thereducing agent is an acid.
 7. The compound of claim 6, wherein the acidis selected from HX, wherein HX is HCl.
 8. The compound of claim 1,wherein compound 10 is produced by converting compound 9

into compound 10


9. The compound of claim 8, wherein compound 9 is converted to compound10 in the presence of a reducing agent and metal catalyst.
 10. Thecompound of claim 9, wherein the reducing agent is ammonium formate andthe metal catalyst is palladium on carbon.
 11. The compound of claim 1,wherein compound 9 is produced by converting compound 8

into compound 9


12. The compound of claim 11, wherein compound 8 is reacted withHNO₃/H₂SO₄ to form a nitrated product.
 13. The compound of claim 12,wherein the nitration product is subjected to potassium hydroxide inmethanol to form compound
 9. 14. The compound of claim 1, whereincompound 8 is produced by converting compound 7

into compound 8


15. The compound of claim 14, wherein the reaction of compound 7 to formcompound 8 is performed in the presence of an acyloxycarbonylating agentand base.
 16. The compound of claim 15, wherein the acyloxycarbonylatingagent is methyl chloroformate and the base is triethylamine.
 17. Thecompound of claim 14, wherein the reaction is performed in the presenceof 4-dimethylaminopyridine.
 18. The compound of claim 1, wherein theprocess further comprises converting tert-butyl phenol

into compound 7


19. The compound of claim 18, wherein the conversion of tert-butylphenol to form the compound of Formula 7 is performed in the presence ofa source of X—C(CD₃)₃ and an acid.
 20. The compound of claim 19, whereinthe source of X—C(CD₃)₃ is tert-butanol-d₁₀ and the acid is D₂SO₄.